Secondary active transporters use the energy from electrochemical ion gradients and/or substrate gradients to mediate concentrative substrate translocation across biological membranes of all organisms. With advances in membrane protein structural biology, it has become clear that a large number of secondary active symporters and exchangers, which belong to distant families without discernable sequence identity, nonetheless, share common structural features that classify them into a single structural family, known as the LeuT-fold. This fold is characterized by 10 transmembrane helices (TMs) organized into two inverted structural repeats each containing 5 TMs. In Bridge 3 we seek to understand commonalities as well as divergence in the functional mechanisms of the LeuT-fold proteins, with a focus on the general rules of protein structural dynamics that underlie function, in the context of differences in their driving mechanism and the conformational changes associated with substrate translocation. To achieve these goals we build on the synergistic approach we have established with the study of LeuT, employing iterative computational, functional and spectroscopic methods. The working hypothesis of this Bridge is that the discovery and interpretation of mechanistic differences between LeuT and other LeuT-fold transporters depends on revealing dynamic properties enabled by local structural differences. We will use a new generation of quantitative computational approaches, in parallel with binding and flux studies, and with EPR and single-molecule fluorescence studies to measure distance between pairs of probes in different conformational states as well as the dynamics of the associated movements. The work will take advantage of specific established collaborations among the team members, and with the Computational Modeling, Spectroscopy and Instrumentation, and Protein Expression Cores. ApcT, a member of the APC family that also includes product/precursor exchangers, shares the LeuT-fold but has been reported to be a H+-dependent amino acid transporter. Interestingly, ApcT has the side chain ?-amino group of Lys158 occupying what is the Na2 site in LeuT, and it has been suggested that protonation and deprotonation of this Lys, like binding and unbinding of Na2 in LeuT, drives transport. In contrast to the profound mechanistic differences between ApcT and LeuT, the Drosophila dopamine transporter (dDAT) is closely related to LeuT in overall structure and function but differentiated by the presence of large amino and carboxy termini, which have been shown to critically modulate transporter function in the eukaryotic transporters. To understand similarities and differences in functional mechanisms for these compared LeuT- fold transporters we propose the following Specific Aims: 1) To determine how substrates, H+ and Na+ coordinate dynamics and conformational changes in the transport cycle of ApcT as compared with LeuT. 2) To integrate CW and DEER measurements in exploring the role of the amino terminus in modulating the conformational dynamics of dDAT.